Identification of CCR4C as a chloroplast-localized NADP(H) phosphatase regulating NAD(P)(H) balance in <i>Arabidopsis</i>

NAD(P)(H) metabolism plays a crucial role in plant development and growth. NADK2, a chloroplast-localized NAD kinase, supplies NADP⁺ to the photosynthetic electron transport chain. The <i>Arabidopsis</i> T-DNA insertion mutant of NADK2 (<i>nadk2</i>) exhibits a reduced NADP⁺/NAD⁺ ratio, slow growth, and pale green leaves. To gain further insights into NAD(P)(H) metabolism in chloroplasts, <i>nadk2</i> revertant mutants (<i>nkr</i>) were screened from the M2 generation of EMS (ethyl methane sulfonate)-treated <i>nadk2</i> seeds. Among them, <i>nkr1</i> displayed greener leaves and improved growth compared to <i>nadk2</i>. Genetic mapping and genomic sequencing identified <i>At3g18500</i> (<i>CCR4C</i>) as the causal gene. The <i>nkr1</i> mutant carried a single nucleotide substitution, introducing a stop codon within the predicted N-terminal chloroplast localization signal, resulting in the loss of CCR4C protein function. The <i>nadk2 ccr4c</i> double mutant restored leaf color and growth to near wild-type levels. To investigate the function of CCR4C, recombinant CCR4C protein was purified and shown to directly convert NADP(H) to NAD(H). Localization analysis with CCR4C-GFP fusion proteins confirmed chloroplast targeting. Furthermore, <i>ccr4c</i> single mutants exhibited disrupted NAD(P)(H) balance and enhanced tolerance to ROS stress (e.g., H₂O₂, methyl viologen). These findings reveal CCR4C as a chloroplast-localized NADP(H) phosphatase crucial for maintaining NAD(P)(H) balance, providing insights into how plant cells manage chloroplast metabolism.